Natural products are usually isolated from free-living terrestrial or aquatic organisms that have no connection with human biology. In contrast, microbes that have evolved to live in close association with humans are especially likely to synthesize natural products that inhibit key human targets, and remain underexplored as a source of novel molecules. We propose a systematic approach to identify natural products from the fungal pathogen Histoplasma capsulatum, which is a soil organism that spends much of its time in the mammalian lung. In terms of identifying new natural products that bind novel human targets, Histoplasma is a particularly compelling choice for the following reasons: (1) It has evolved to live within human immune cells. Although other fungi are capable of colonizing immunocompromised hosts, very few can colonize immunocompetent hosts (a more difficult task that indicates that H. capsulatum can manipulate the normal innate immune response). (2) It is straightforward to grow production-scale cultures of Histoplasma for natural product isolation. (3) Histoplasma molecular genetics allows the straightforward construction of mutants that lack putative biosynthetic enzymes. (4) Although analysis of the genome predicts that each strain of Histoplasma produces 10-20 natural products, neither Histoplasma nor any of its close relatives have ever been mined for novel molecules by an academic or industrial natural product discovery effort. In sum, since Histoplasma is an intracellular pathogen that subverts the innate immune response to colonize host immune cells by unknown mechanisms, we hypothesize that Histoplasma natural products may be enriched for novel compounds that modulate targets in mammalian hosts. We will combine the expertise of the Sil lab in Histoplasma biology and molecular genetics with the expertise of the Fischbach lab in natural product discovery to comprehensively identify and characterize natural products secreted by Histoplasma. We propose a three-pronged approach: (1) Based on our preliminary data, we will take a microbial approach, growing large quantities of Histoplasma under laboratory conditions that recapitulate its free-living and host-associated environments. Utilizing standard expertise in the Fischbach lab, we will purify milligram quantities of 4-6 abundant natural products by preparative HPLC, and use high-resolution MS and NMR to solve their structures. (2) Taking advantage of expertise in the Sil lab, we will take a complementary genetic approach by using RNA interference to deplete Histoplasma cells of individual natural product biosynthetic genes. Both predictive algorithms from the Fischbach lab and the resultant changes in the HPLC/MS profile in supernatant extracts from the mutant strains will allow us to correlate particular molecules with their biosynthetic genes. (3) We will execute a series of in vitro and in vivo assays to determine the bioactivity of natural products identified by this project, with an emphasis on the identification of biosynthetic genes and natural products that contribute to disease pathogenesis. These exploratory studies of natural product biology in a ubiquitous, human-associated microbe have strong potential for uncovering new molecules that modulate communication at the host-pathogen interface.